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S6K1/2 | Ribosomal S6 kinase 1/2

265 €
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AS12 1855 | clonality: polyclonal | host: rabbit | reactivity: Arabidopsis thaliana

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Item No:
AS12 1855

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product information
Background

Ribosomal S6 kinase 1/2 (S6K1/2) involved in TOR signaling pathway, in osmotic stress response. Activated by PDK1 and repressed during osmotic stress. Expressed in all tissues, especially during high metabolic activity in growing buds, root tips, leaf margins and germinating seeds. Alternative names: AtPK1/AtPK6 S6K1), AtPK2/AtPK19 (S6K2).

Immunogen

KLH-conjugated peptide, derived from Arabidopsis thaliana S6K1: UniProt: P42818, TAIR: AT3G08730 and S6K2: UniProt: Q39030, TAIR: AT3G08720. Due to high amino acid homology, chosen peptide is conserved in both proteins:  S6K1 and S6K2.

Host Rabbit
Clonality Polyclonal
Clone
Purity Affinity purified serum in PBS, pH 7.4
Format Liquid in PBS pH 7.4 and 0.02% sodium azide as preservative
Quantity 50 ĩg
Reconstitution
Storage Store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles.Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from lyophilized material adhering to the cap or sides of the tubes.
Tested applications Western blot (WB)
Related products

AS12 2608 | TOR | Target of rapamycin, rabbit antibody
AS13 2664 | S6K1-2 | Ribosomal-protein S6 kinase homolog 1,2 - phosphorylated, rabbit antibody

Plant protein extraction buffer

Secondary antibodies

Additional information So far in applied conditions and extracts endogenous S6K1/2 protein is detectable as a very weak band.
application information
Recommended dilution 1 : 750-1 : 1000 (WB)
Expected | apparent MW

52.6 kDa (S6K1) and 53 kDa (S6K2)

Confirmed reactivity Arabidopsis thaliana
Predicted reactivity Thelungiella halophila
Not reactive in Setaria viridis
Additional information Keeping the samples at 4°C all times is of crucial importance. Laucs buffer was used in example below since this is the one which was used routinely by this test laboratory for the work with kinases. Some unspecific bands can be seen, depending upon western blot protocol which is used. Therefore please consider to use a negative control together with your samples.
Selected references Pfeiffer et al. (2016). Integration of light and metabolic signals for stem cell activation at the shoot apical meristem. Elife. 2016 Jul 11;5. pii: e17023. doi: 10.7554/eLife.17023.

application example

western blot using anti-S6K/2 antibodies


20 µg of total protein from flowers and leaves of transgenic Arabidopsis thaliana lines were analysed (expressing the genomic copy of S6K1 tagged with FLAG epitop under the control of its own promoter) extracted with homogenization buffer were separated on 10% SDS-PAGE and blotted 2h to PVDF. Blots were blocked with 5% milk in TBST for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1:750 for overnight at 4C with agitation. The antibody solution was decanted and the blot was washed three times for 15 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated from Agrisera, AS09 602) diluted to 1:10 000 in 2.5% milk in TBST for 1h at RT with agitation. The blot was washed as above and developed for 5 min with ECL according to the manufacturer's instructions (PIERCE). Exposure time was few minutes.

Courtesy of Dr. Rossana Henriques, CRAG, Spain


western blot using anti-S6K polyclonal antibodies

20 µg of total protein from Arabidopsis thaliana total wilde type (Col-0), deletion mutant (s6k1-1), overexpression mutant (S6K1g-CFP) extracted with homogenization buffer were separated on 10% SDS-PAGE and blotted 2h to PVDF. Blots were blocked with 5% milk in TBST for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1:1000 for overnight at 4C with agitation. The antibody solution was decanted and the blot was washed three times for 15 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated from Agrisera, AS09 602) diluted to 1:10 000 in 2.5% milk in TBST for 1h at RT with agitation. The blot was washed as above and developed for 5 min with ECL according to the manufacturer's instructions (PIERCE). Exposure time was few minutes.

Courtesy of Dr. Rossana Henriques, CRAG, Spain

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